Combination vaccine

ABSTRACT

The disclosure relates to a composition comprising two or more immunogenic staphylococcal polypeptides and a multivalent vaccine composition comprising the immunogenic staphylococcal polypeptides.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 100193_401D1_SEQUENCE_LISTING.txt. The text fileis 40.1 KB, was created on Dec. 14, 2015, and is being submittedelectronically via EFS-Web.

TECHNICAL FIELD

The disclosure relates to a composition comprising two or moreimmunogenic staphylococcal polypeptides and a multivalent vaccinecomposition comprising the immunogenic staphylococcal polypeptides inthe prevention or treatment of staphylococcal infections in humans andanimals.

BACKGROUND

Vaccines protect against a wide variety of infectious diseases. Manymodern vaccines are therefore made from protective antigens of thepathogen, which are isolated by molecular cloning and purified. Thesevaccines are known as ‘subunit vaccines’. The development of subunitvaccines has been the focus of considerable research in recent years.The emergence of new pathogens and the growth of antibiotic resistancehave created a need to develop new vaccines and to identify furthercandidate molecules useful in the development of subunit vaccines.Likewise the discovery of novel vaccine antigens from genomic andproteomic studies is enabling the development of new subunit vaccinecandidates, particularly against bacterial pathogens. However, althoughsubunit vaccines tend to avoid the side effects of killed or attenuatedpathogen vaccines, their ‘pure’ status means that subunit vaccines donot always have adequate immunogenicity to confer protection.

An approach to improve the efficacy of vaccine compositions is toprovide multivalent vaccines comprising dominant antigens that provokeboth a B cell and T cell response thereby mounting a more rigorousimmune response in the subject receiving the vaccine. A typicalmultivalent vaccine might be a whole cell vaccine comprising multipleantigenic molecules. For example the Bacillus Calmette Guerin [“BCG”]vaccine includes an attenuated Mycobacterium bovis strain that provokesprotective immunity in humans. For many pathogens chemical or heatinactivation while it may give rise to vaccine immunogens that conferprotective immunity also gives rise to side effects such as fever andinjection site reactions. In the case of bacteria, inactivated organismstend to be so toxic that side effects have limited the application ofsuch crude vaccine immunogens and therefore vaccine development haslagged behind drug-development. Moreover, effective vaccine developmentusing whole cell inactivated organisms suffers from problems of epitopemasking, immunodominance, low antigen concentration and antigenredundancy.

SUMMARY

Currently there is no effective vaccination procedure to prevent ortreat Staphylococcus aureus infection. S. aureus is a bacterium whosenormal habitat is the epithelial lining of the nose in about 20-40% ofnormal healthy people and is also commonly found on people's skinusually without causing harm. However, in certain circumstances,particularly when skin is damaged, this pathogen can cause infection.This is a particular problem in hospitals where patients may havesurgical procedures and/or be taking immunosuppressive drugs. Thesepatients are much more vulnerable to infection with S. aureus because ofthe treatment they have received. Antibiotic resistant strains of S.aureus have arisen since their wide spread use in controlling microbialinfection. Methicillin resistant strains are prevalent and many of theseresistant strains are also resistant to several other antibiotics.

S. aureus is therefore a major human pathogen capable of causing a widerange of diseases some of which are life threatening diseases includingsepticaemia, endocarditis, arthritis and toxic shock. This ability isdetermined by the versatility of the organism and its arsenal ofcomponents involved in virulence. At the onset of infection, and as itprogresses, the needs and environment of the organism changes and thisis mirrored by a corresponding alteration in the virulence determinantswhich S. aureus produces. At the beginning of infection it is importantfor the pathogen to adhere to host tissues and so a large repertoire ofcell surface associated attachment proteins are made. The pathogen alsohas the ability to evade host defences by the production of factors thatreduce phagocytosis or interfere with the ability of the cells to berecognised by circulating antibodies.

There is therefore a continuing need to identify staphylococcal antigensthat are protective and can be used in multivalent vaccines. Thecombinations may be used in combination with non-protein immunogenicmolecules such as polysaccharide antigens and anti-bacterial agents toprovide a treatment regimen for control of staphylococcal infection. Itis also within the scope of this disclosure to modify the treatmentregimen to immunize subjects with a series of temporally separatedadministrations as an alternative to the administration of a singlevaccine comprising multiple antigens.

This disclosure therefore relates to combination or multivalentimmunogenic compositions and vaccines and their use in the prophylaxisand treatment of staphylococcal infections. We disclose polypeptidesthat individually are protective and are typically membrane spanningproteins that include an extracellular domain and are essential forstaphylococcal cell growth. For example DivIB is an integral membraneprotein comprising an intracellular domain, an intermembrane domain andan extracellular domain. DivIB and fragments thereof, provide protectionfrom at least an S. aureus challenge in an animal model. The relatedgene DivIC is also an integral membrane protein the extracellular domainof which provokes protective immunity to staphylococcal infection. Thisdisclosure also relates to antigens encoded by the genes PheP, YdiE andFtsL, each of which has an extramembranous domain.

According to an aspect of the invention there is provided an immunogeniccomposition comprising two or more different polypeptides wherein saidpolypeptides are encoded by different staphylococcal genes selected fromthe group consisting of:

-   -   i) a polypeptide, or immunogenic fragment thereof, comprising or        consisting of the amino acid sequence as represented in SEQ ID        NO.: 20;    -   ii) a polypeptide, or immunogenic fragment thereof, comprising        or consisting of the amino acid sequence as represented in SEQ        ID NO.: 21    -   iii) a polypeptide, or immunogenic fragment thereof, comprising        or consisting of the amino acid sequence as represented in SEQ        ID NO.: 22;    -   iv) a polypeptide, or immunogenic fragment thereof, comprising        or consisting of the amino acid sequence as represented in SEQ        ID NO.: 23;    -   v) a polypeptide, or immunogenic fragment thereof, comprising or        consisting of the amino acid sequence as represented in SEQ ID        NO.: 24; or    -   vi) a modified staphylococcal polypeptide wherein said        polypeptide is a staphylococcal polypeptide variant of the amino        acid sequences presented in SEQ ID NO.: 20, 21, 22, 23 or 24,        wherein said sequences are modified by addition, deletion or        substitution of one or more amino acid residues which modified        polypeptides have retained or enhanced immunogenicity when        compared to the polypeptide as represented in SEQ ID NO.: 20,        21, 22, 23 or 24.

A modified staphylococcal polypeptide or variant staphylococcalpolypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions, truncations that may be present inany combination. Among preferred variants are those that vary from areference polypeptide by conservative amino acid substitutions. Suchsubstitutions are those that substitute a given amino acid by anotheramino acid of like characteristics. The following non-limiting list ofamino acids are considered conservative replacements (similar): a)alanine, serine, and threonine; b) glutamic acid and aspartic acid; c)asparagine and glutamine d) arginine and lysine; e) isoleucine, leucine,methionine and valine and f) phenylalanine, tyrosine and tryptophan.Most highly preferred are variants that retain or enhance theimmunogenicity and/or activity as the reference polypeptide from whichit varies.

In one embodiment, the variant polypeptides have at least 80-89%sequence identity, more preferably at least 90% identity, even morepreferably at least 95% identity, still more preferably at least 97%identity, and most preferably at least 99% identity with the full lengthamino acid sequences illustrated herein.

In a preferred embodiment of the invention said immunogenic compositioncomprises or consists essentially of 2, 3, 4 or 5 staphylococcalpolypeptides.

In a preferred embodiment of the invention there is provided animmunogenic composition comprising:

-   -   i) a polypeptide comprising SEQ ID NO.: 20, or an antigenic        fragment thereof; and    -   ii) a polypeptide comprising SEQ ID NO.: 21, or antigenic        fragment thereof.

In a preferred embodiment of the invention there is provided animmunogenic composition comprising:

-   -   i) a polypeptide comprising SEQ ID NO.: 20, or an antigenic        fragment thereof; and    -   ii) a polypeptide comprising SEQ ID NO.: 22, or an antigenic        fragment thereof.

In a preferred embodiment of the invention there is provided animmunogenic composition comprising:

-   -   i) a polypeptide comprising SEQ ID NO.:20, or an antigenic        fragment thereof; and    -   ii) a polypeptide comprising SEQ ID NO.:23, or an antigenic        fragment thereof.

In a preferred embodiment of the invention there is provided acomposition comprising:

-   -   i) a polypeptide comprising SEQ ID NO.: 20, or an antigenic        fragment thereof; and    -   ii) a polypeptide comprising SEQ ID NO.: 24, or an antigenic        fragment thereof.

In a preferred embodiment of the invention there is provided animmunogenic composition comprising:

-   -   i) a polypeptide comprising SEQ ID NO.: 20, or an antigenic        fragment thereof; and    -   ii) a polypeptide comprising SEQ ID NO.: 23 and 24, or an        antigenic fragment thereof.

In a preferred embodiment of the invention there is provided animmunogenic composition comprising:

-   -   i) a polypeptide comprising SEQ ID NO.: 20, or an antigenic        fragment thereof; and    -   ii) a polypeptide comprising SEQ ID NO.: 21 and 22, or an        antigenic fragment thereof.

In a preferred embodiment of the invention there is provided animmunogenic composition comprising:

-   -   i) a polypeptide comprising SEQ ID NO.: 20, or an antigenic        fragment thereof; and    -   ii) a polypeptide comprising SEQ ID NO.: 22 and 23, or an        antigenic fragment thereof.

In a preferred embodiment of the invention there is provided animmunogenic composition comprising:

-   -   i) a polypeptide comprising SEQ ID NO.: 20, or an antigenic        fragment thereof; and    -   ii) a polypeptide comprising SEQ ID NO.: 21 and 23, or an        antigenic fragment thereof.

In a preferred embodiment of the invention there is provided animmunogenic composition comprising:

-   -   i) a polypeptide comprising SEQ ID NO.: 20, or an antigenic        fragment thereof; and    -   ii) a polypeptide comprising SEQ ID NO.: 22 and 24, or an        antigenic fragment thereof.

In a preferred embodiment of the invention there is provided animmunogenic composition comprising:

-   -   i) a polypeptide comprising SEQ ID NO.: 20, or an antigenic        fragment thereof; and    -   ii) a polypeptide comprising SEQ ID NO.: 21 and 24, or an        antigenic fragment thereof.

In a preferred embodiment of the invention there is provided animmunogenic composition comprising:

-   -   i) a polypeptide comprising SEQ ID NO.: 20, or an antigenic        fragment thereof; and    -   ii) a polypeptide comprising SEQ ID NO.: 21, 23 and 24, or an        antigenic fragment thereof.

In a preferred embodiment of the invention there is provided animmunogenic composition comprising:

-   -   i) a polypeptide comprising SEQ ID NO.: 20, or an antigenic        fragment thereof; and    -   ii) a polypeptide comprising SEQ ID NO.: 22, 23 and 24, or an        antigenic fragment thereof.

In a preferred embodiment of the invention there is provided animmunogenic composition comprising:

-   -   i) a polypeptide comprising SEQ ID NO.: 20, or an antigenic        fragment thereof; and    -   ii) a polypeptide comprising SEQ ID NO.: 21, 22 and 24, or an        antigenic fragment thereof.

In a preferred embodiment of the invention there is provided animmunogenic composition comprising:

-   -   i) a polypeptide comprising SEQ ID NO.: 20, or an antigenic        fragment thereof; and    -   ii) a polypeptide comprising SEQ ID NO.: 21, 22 and 23, or an        antigenic fragment thereof.

In a preferred embodiment of the invention there is provided animmunogenic composition comprising:

-   -   i) a polypeptide comprising SEQ ID NO.: 20, or an antigenic        fragment thereof; and    -   ii) a polypeptide comprising SEQ ID NO.: 21, 22, 23 and 24, or        an antigenic fragment thereof.

In an alternative preferred embodiment of the invention there isprovided an immunogenic composition comprising:

-   -   i) a polypeptide comprising SEQ ID NO.: 21, or an antigenic        fragment thereof; and    -   ii) a polypeptide comprising SEQ ID NO.: 22, or an antigenic        fragment thereof.

In a preferred embodiment of the invention there is provided animmunogenic composition comprising:

-   -   i) a polypeptide comprising SEQ ID NO.: 21, or an antigenic        fragment thereof; and    -   ii) a polypeptide comprising SEQ ID NO.: 23, or an antigenic        fragment thereof.

In a preferred embodiment of the invention there is provided animmunogenic composition comprising:

-   -   i) a polypeptide comprising SEQ ID NO.: 21, or an antigenic        fragment thereof; and    -   ii) a polypeptide comprising SEQ ID NO.: 24, or an antigenic        fragment thereof.

In a preferred embodiment of the invention there is provided animmunogenic composition comprising:

-   -   i) a polypeptide comprising SEQ ID NO.: 21, or an antigenic        fragment thereof; and    -   ii) a polypeptide comprising 23 and 24, or an antigenic fragment        thereof.

In a preferred embodiment of the invention there is provided animmunogenic composition comprising:

-   -   i) a polypeptide comprising SEQ ID NO.: 21, or an antigenic        fragment thereof; and    -   ii) a polypeptide comprising SEQ ID NO.: 22, and 23, or an        antigenic fragment thereof.

In a preferred embodiment of the invention there is provided animmunogenic composition comprising:

-   -   i) a polypeptide comprising SEQ ID NO.: 21, or an antigenic        fragment thereof; and    -   ii) a polypeptide comprising SEQ ID NO.: 22 and 24, or an        antigenic fragment thereof.

In a preferred embodiment of the invention there is provided animmunogenic composition comprising:

-   -   i) a polypeptide comprising SEQ ID NO.: 21, or an antigenic        fragment thereof; and    -   ii) a polypeptide comprising SEQ ID NO.: 22, 23 and 24, or an        antigenic fragment thereof.

In an alternative preferred embodiment of the invention there isprovided an immunogenic composition comprising:

-   -   i) a polypeptide comprising SEQ ID NO.: 22, or an antigenic        fragment thereof; and    -   ii) a polypeptide comprising SEQ ID NO.: 23 and 24, or an        antigenic fragment thereof.

In a preferred embodiment of the invention there is provided animmunogenic composition comprising:

-   -   i) a polypeptide comprising SEQ ID NO.: 22, or an antigenic        fragment thereof; and    -   ii) a polypeptide comprising SEQ ID NO.: 23, or an antigenic        fragment thereof.

In a preferred embodiment of the invention there is provided animmunogenic composition comprising:

-   -   i) a polypeptide comprising SEQ ID NO.: 22, or an antigenic        fragment thereof; and    -   ii) a polypeptide comprising SEQ ID NO.: 24, or an antigenic        fragment thereof.

In an alternative preferred embodiment of the invention there isprovided an immunogenic composition comprising:

-   -   i) a polypeptide comprising SEQ ID NO.: 23, or an antigenic        fragment thereof; and    -   ii) a polypeptide comprising SEQ ID NO.: 24, or an antigenic        fragment thereof.

DETAILED DESCRIPTION

In a preferred embodiment of the invention said composition is a vaccinecomposition and includes at least one carrier and/or adjuvant.

Adjuvants (immune potentiators or immunomodulators) have been used fordecades to improve the immune response to vaccine antigens. Theincorporation of adjuvants into vaccine formulations is aimed atenhancing, accelerating and prolonging the specific immune response tovaccine antigens. Advantages of adjuvants include the enhancement of theimmunogenicity of weaker antigens, the reduction of the antigen amountneeded for a successful immunisation, the reduction of the frequency ofbooster immunisations needed and an improved immune response in elderlyand immunocompromised vaccines. Selectively, adjuvants can also beemployed to optimise a desired immune response, e.g. with respect toimmunoglobulin classes and induction of cytotoxic or helper T lymphocyteresponses. In addition, certain adjuvants can be used to promoteantibody responses at mucosal surfaces. Aluminium hydroxide andaluminium or calcium phosphate has been used routinely in humanvaccines.

Adjuvants can be classified according to their source, mechanism ofaction and physical or chemical properties. The most commonly describedadjuvant classes are gel-type, microbial, oil-emulsion andemulsifier-based, particulate, synthetic and cytokines. More than oneadjuvant may be present in the final vaccine product according to theinvention. They may be combined together with a single antigen or allantigens present in the vaccine, or each adjuvant may be combined withone particular antigen. The origin and nature of the adjuvants currentlybeing used or developed is highly diverse. For example, aluminium basedadjuvants consist of simple inorganic compounds and PLG is a polymericcarbohydrate. MDP is derived from bacterial cell walls; saponins are ofplant origin, squalene is derived from shark liver and recombinantendogenous immunomodulators are derived from recombinant bacterial,yeast or mammalian cells. There are several adjuvants licensed forveterinary vaccines, such as mineral oil emulsions that are too reactivefor human use. Similarly, complete Freund's adjuvant, although being oneof the most powerful adjuvants known, is not suitable for human use.

A carrier is an immunogenic molecule which, when bound to a secondmolecule augments immune responses to the latter. The term carrier isconstrued in the following manner. A carrier is an immunogenic moleculewhich, when bound to a second molecule augments immune responses to thelatter. Some antigens are not intrinsically immunogenic yet may becapable of generating antibody responses when associated with a foreignprotein molecule such as keyhole-limpet haemocyanin or tetanus toxoid.Such antigens contain B-cell epitopes but no T cell epitopes. Theprotein moiety of such a conjugate (the “carrier” protein) providesT-cell epitopes which stimulate helper T-cells that in turn stimulateantigen-specific B-cells to differentiate into plasma cells and produceantibody against the antigen.

The vaccine compositions of the invention can be administered by anyconventional route, including injection, intranasal spray by inhalationof for example an aerosol or nasal drops. The administration may be, forexample, intravenous, intraperitoneal, intramuscular, intracavity,subcutaneous, or intradermally. The vaccine compositions of theinvention are administered in effective amounts. An “effective amount”is that amount of a vaccine composition that alone or together withfurther doses, produces the desired response. In the case of treating aparticular bacterial disease the desired response is providingprotection when challenged by an infective agent.

In a preferred embodiment of the invention said vaccine composition isadapted for administration as a nasal spray.

In a preferred embodiment of the invention said vaccine composition isprovided in an inhaler and delivered as an aerosol.

The amounts of vaccine will depend, of course, on the individual patientparameters including age, physical condition, size and weight, theduration of the treatment, the nature of concurrent therapy (if any),the specific route of administration and like factors within theknowledge and expertise of the health practitioner. These factors arewell known to those of ordinary skill in the art and can be addressedwith no more than routine experimentation. It is generally preferredthat a maximum dose of the individual components or combinations thereofbe used sufficient to provoke immunity; that is, the highest safe doseaccording to sound medical judgment. It will be understood by those ofordinary skill in the art, however, that a patient may insist upon alower dose or tolerable dose for medical reasons, psychological reasonsor for virtually any other reasons.

The doses of vaccine administered to a subject can be chosen inaccordance with different parameters, in particular in accordance withthe mode of administration used and the state of the subject. In theevent that a response in a subject is insufficient at the initial dosesapplied, higher doses (or effectively higher doses by a different, morelocalized delivery route) may be employed to the extent that patienttolerance permits.

In general, doses of vaccine are formulated and administered ineffective immunizing doses according to any standard procedure in theart. Other protocols for the administration of the vaccine compositionswill be known to one of ordinary skill in the art, in which the doseamount, schedule of injections, sites of injections, mode ofadministration and the like vary from the foregoing. Administration ofthe vaccine compositions to mammals other than humans, (e.g. for testingpurposes or veterinary therapeutic purposes), is carried out undersubstantially the same conditions as described above. A subject, as usedherein, is a mammal, preferably a human, and including a non-humanprimate, cow, horse, pig, sheep or goat.

In a preferred embodiment of the invention there is provided a vaccinecomposition according to the invention that includes at least oneadditional anti-bacterial agent.

In a preferred embodiment of the invention said agent is a seconddifferent vaccine and/or immunogenic agent (for example a bacterialpolypeptide and/or polysaccharide antigen).

According to a further aspect of the invention there is provided acomposition comprising a nucleic acid molecule[s] comprising orconsisting of nucleotide sequences of two or more differentstaphylococcal genes and encoding immunogenic polypeptides selected fromthe group consisting of:

-   -   i) a nucleic acid molecule comprising or consisting of the        nucleotide sequence as represented in SEQ ID NO.: 1 or 6;    -   ii) a nucleic acid molecule comprising or consisting of the        nucleotide sequence as represented in SEQ ID NO.: 2 or 7;    -   iii) a nucleic acid molecule comprising or consisting of the        nucleotide sequence as represented in SEQ ID NO.: 3 or 8;    -   iv) a nucleic acid molecule comprising or consisting of the        nucleotide sequence as represented in SEQ ID NO.: 4 or 9;    -   v) a nucleic acid molecule comprising or consisting of the        nucleotide sequence as represented in SEQ ID NO.: 5 or 10;    -   vi) a nucleic acid molecule comprising or consisting of a        nucleotide sequence wherein said sequence is degenerate as a        result of the genetic code to the nucleotide sequence defined in        i-v above; or    -   vii) a nucleic acid molecule the complementary strand of which        hybridizes under stringent hybridization conditions to the        sequence in SEQ ID NO.: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and        wherein said nucleic acid molecule encodes a staphylococcal        antigenic polypeptide.

Hybridization of a nucleic acid molecule occurs when two complementarynucleic acid molecules undergo an amount of hydrogen bonding to eachother. The stringency of hybridization can vary according to theenvironmental conditions surrounding the nucleic acids, the nature ofthe hybridization method, and the composition and length of the nucleicacid molecules used. Calculations regarding hybridization conditionsrequired for attaining particular degrees of stringency are discussed inSambrook et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N. Y., 2001); and Tijssen,Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes Part I, Chapter 2(Elsevier, New York, 1993). The T, is the temperature at which 50% of agiven strand of a nucleic acid molecule is hybridized to itscomplementary strand. The following is an exemplary set of hybridizationconditions and is not limiting:

Very High Stringency (Allows Sequences that Share at Least 90% Identityto Hybridize)

Hybridization: 5×SSC at 65° C. for 16 hours

Wash twice: 2×SSC at room temperature (RT) for 15 minutes each

Wash twice: 0.5×SSC at 65° C. for 20 minutes each

High Stringency (Allows Sequences that Share at Least 80% Identity toHybridize)

Hybridization: 5×-6×SSC at 65° C.-70° C. for 16-20 hours

Wash twice: 2×SSC at RT for 5-20 minutes each

Wash twice: 1×SSC at 55° C.-70° C. for 30 minutes each

Low Stringency (Allows Sequences that Share at Least 50% Identity toHybridize)

Hybridization: 6×SSC at RT to 55° C. for 16-20 hours

Wash at least twice: 2×-3×SSC at RT to 55° C. for 20-30 minutes each.

In a preferred embodiment of the invention said composition is a vaccinecomposition and includes at least one carrier and/or adjuvant.

The nucleic acid or DNA combination vaccines comprise nucleic acidmolecules that encode antigenic polypeptides as herein disclosed. Thespecific combinations of polypeptide antigens as represented by aminoacid SEQ ID can be substituted for the corresponding nucleotide SEQ IDas herein disclosed in the manufacture of DNA vaccines.

According to a further aspect of the invention there is provided acombination vaccine according to the invention for use in the protectionor treatment of a subject animal to a staphylococcal infection orcondition that results from a staphylococcal infection.

In a preferred embodiment of the invention said staphylococcal infectionis caused by a staphylococcal species selected from the group consistingof: S. epidermidis, S. aureus, S. hominis, S. haemolyticus, S. wameri,S. capitis, S. saccharolyticus, S. auricularis, S. simulans, S.saprophyticus, S. cohnii, S. xylosus, S. hyicus, S. caprae, S.gallinarum, and S. intermedius.

In a further preferred embodiment of the invention said staphylococcalspecies is S. aureus or S. epidermidis.

In a preferred embodiment of the invention said subject is a human.

In an alternative preferred embodiment of the invention said subject isa non-human animal, preferably a livestock animal, for example cattle.

In a preferred embodiment of the invention said live stock animal isvaccinated against bacterial mastitis caused by staphylococcal bacterialcells.

In a preferred embodiment of the invention said life stock animal is acaprine animal (e.g. sheep, goat).

In a preferred embodiment of the invention said life stock animal is abovine animal (e.g. a cow).

Staphylococcal mastitis is a serious condition that affects live stockand can result in considerable expense with respect to controlling thedisease through administration of antibiotics and in terms of lost milkyield. The vaccine according to the invention provides cost effectivecontrol of bacterial, in particular staphylococcal mastitis.

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of the words, for example“comprising” and “comprises”, means “including but not limited to”, andis not intended to (and does not) exclude other moieties, additives,components, integers or steps.

Throughout the description and claims of this specification, thesingular encompasses the plural unless the context otherwise requires.In particular, where the indefinite article is used, the specificationis to be understood as contemplating plurality as well as singularity,unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties orgroups described in conjunction with a particular aspect, embodiment orexample of the invention are to be understood to be applicable to anyother aspect, embodiment or example described herein unless incompatibletherewith.

An embodiment of the invention will now be described by example only, asdescribed herein.

Materials and Methods

Construction of Plasmids for the Overexpression in E. coli of theExtramembranous Fragments of the S. aureus Proteins

The PheP selected peptide was synthesized and conjugated through acysteine at its C terminal to the carrier protein KLH to undertake as achimeric protein used in vaccinations. The extramembranous fragments ofYdiE, DivIB, DivIC and FtsL were PCR amplified from the chromosome ofstrain S. aureus SH1000 (Horsburgh M J, Aish J L, White I J, Shaw L,Lithgow J K, Foster S J: sigmaB modulates virulence determinantexpression and stress resistance: characterization of a functional rsbUstrain derived from Staphylococcus aureus 8325-4. J. Bacteriol.184:5457-5467, 2002) using oligonucleotide pairs indicated on Table 1according to the following PCR reaction conditions: 1 initialdenaturation cycle of 94° C. for 4 min; 30 amplification cycles ofdenaturation 94° C. for 30 seconds, annealing 45° C. for 30 seconds, andextension at 72° C. for up to 2.5 minutes; finally, ongoingamplification rounds were allow to complete at 72° C. for 4 min.

The restrictions sites engineered within the oligonucleotides are alsoindicated in Table 1 (underlined; Ncol or Xhol). The amplified fragmentswere digested with the corresponding restriction enzymes (Ncol for the5′ end, and Xhol for the 3′ end) and cloned into the equivalent sites ofthe pET-21d(+) expression vector from Novagen (Cat. No. 69743-3) andresulting in the overexpression plasmids indicated in Table 1 generatinga T7-tagged (partial, at the N-terminal) and 6×His-tagged (at theC-terminal end) form of the extramembranous fragments. In the SEQ IDsthe T7- and His-tags are indicated in bold, and the extramembranousportion of the proteins of interest are underlined. The over expressionplasmids were transferred into E. coli BL21 for over expression of therecombinant protein fragment.

The cloning of the PCR amplified fragment indicated above into therecipient pET21d(+) recipient plasmid vector at the Ncol and Xhol sitesentailed the addition of hinge amino acids between the T7-tag and theextramembranous fragment, and between the latter and the His-tag. Theseamino acids are neither bold nor underlined in the SEQ IDs.

Over Expression of SEQ ID NOS.: 25-28

SEQ ID NOS.: 25 through 28 were over expressed from plasmids pGL597,pGL601, pALB26, and pALB27 in E. coli BL21 strain using Brain HeartInfusion Broth (CMO225, Oxoid, United Kingdom) in the presence of 100μg/ml ampicillin and the Plac promoter gratuitous inducer IPTG(Isopropyl β-D-1-thiogalactopyranoside, 1 mM) for 4 to 6 hours at 37° C.and vigorous shaking. Following harvesting of the cells bycentrifugation (5,000×g for 15 minutes at 4° C.) and subsequent lysiswith 1 mg/ml lysozyme in phosphate buffer (Buffer A; 0.1M pH7.2)containing 0.5M NaCl) for one hour and subsequent sonication (3 cyclesof 10 second pulses in sonicating water bath) the soluble and insolubleforms of the proteins of interest were separated by centrifugation at13,000×g for 10 minutes. The precipitate was then resuspended in BufferA containing 8M urea by freeze/thawing (3 cycles of freezing at −80° C.for 10 minutes and subsequent thawing to room temperature) andsonication (3 cycles of 10 second pulses in sonicating water bath), andsubsequent centrifugation for 25 minutes at 18,000×g). The overexpressed proteins of interest in the supernatant and the solubilisedpellet were purified by initial specific binding (through their His-tag)to a nickel (NiSO4)-bound Sepharose chromatography column (Ni-Sepharose)and elution with an imidazole solution run through the column in thefollowing stepwise manner: 5% for 5 minutes, 30% for 60 minutes, 35% for60 minutes, 50% for 100 minutes and 55% for 100 minutes. Fractions fromthis stepwise elution were analysed in acrylamide denaturing gels with a4% acrylamide/bis-acrilamide stacking layer and a 12%acrylamide/bis-acrylamide separating layer. The fractions containing theover expressed proteins of interest were pooled and dyalized againststerile phosphate buffer (8 g NaCl, 0.2 g KCl, 1.44 g Na₂HPO₄, 0.24 gKH₂PO₄, per liter of distilled H₂O, pH 7.4).

All the proteins of interest were successfully over expressed from theindicated strains and under the indicated conditions. They were alsosubsequently extracted from the total cellular protein content of theover expressing E. coli strains with more than 95% purity. Examples ofthe purification obtained for each of the proteins are indicated below.

Evaluation of Vaccination-Mediated Protection of Balb/C Mice AgainstInfection by S. aureus

One week after the second boost each animal was infected with an i.v.(tail vein) injection of 100 microliters of endotoxin-free PBScontaining 1.1×10⁷ (±0.5×10⁷) cells of S. aureus strain Newman. Thelatter were prepared from cultures growing to early stationary phase inBrain Heart Infusion medium (BHI), which were then washed three timeswith the same volume of PBS.

After 10 to 14 days the animals were sacrificed according to Schedule 1cervical dislocation. The pair of kidneys from each animal was extractedin aseptic conditions, and homogenized in sterile PBS. Serial dilutionsof the kidney homogenates were carried out in PBS and plated on BHI agarplates. Plates containing between 10 to 150 staphylococcal colonies werecounted and dilution corrected. The number of viable cells in thekidneys was inferred from the number of colony forming units (CFU) onthe plates. Evaluation of the possible protection against infectionconferred by vaccination with DivIB-2 was determined from difference inthe number of S. aureus cells in the kidneys of animals vaccinated withKLH and those vaccinated with DivIB-2. The statistic significance of thedifference was calculated using the Mann-Whitney test. A significantlyhigher (p<0.05) number of S. aureus in KLH vaccinated animals comparedto the DivIB-2 vaccinated animals was concluded as protection.

Vaccination: Generic Protocol for Polyvalent Vaccines

Combination (or polyvalent) vaccines including variations of theantigens (conjugated selected PheP peptide, YdiE, DivIB, DivIC and FtsL)will follow an identical protocol with the following modifications. Thevaccine priming and boost mixtures will contain rather than a singlecomponent 2 or more of the components. The total volume of mixed vaccineused for priming and boosting injections will fluctuate in a range of50-100 microliters per animal. Similarly the total amount in each ofthose injections may vary between 50-100 micrograms. The amount of eachantigen to contribute to the total amount of vaccine in the priming orboosting mix will vary between 20% to 80% of the total.

The various combinations of antigens to be evaluated as a vaccine mixwill be undertaken according to the matrix in Table 2. The combinationsare grouped in 3 tiers. Depending on results from the 1^(st) Tier ofexperiments the 2^(nd) Tier of experiments would be undertakenaccordingly, and depending on the results from the latter the 3^(rd)Tier will be undertaken. In each Tier a vaccination experiment willcontain an antigen in the Y axis, together with those ticked along the Xaxis, and labelled with the same colour. Each vaccination experiment islabelled with a different colour.

Example 1

The experimental sample consisted in a combination antigen of theextracellular domains of YdiE and DivIB. The amount of antigenadministered to each mouse (Female Balb/C, approx. 5-6 weeks old) was 5ug of YdiE plus 50 ug of DivIB. Those amounts were contained within 100ul of eluent consisting on a 50:50 v:v of PBS (Phosphate Buffer Saline)and Complete Freund's Adjuvant (used for the vaccination priming) orIncomplete Freund's Adjuvant (used for the vaccination boost). Primingwas undertaken day 0, Boost 1 at 14 days, and Boost 2 at 21 days.Subsequently, 7 days later, i.e., at day 28 the animals were infectedwith Staphylococcus aureus strain Newman. Each test group (control andexperimental) had 10 animals. The bacterial dose administered to theanimals (both, control and experimental) contained 4×10⁶ bacteria in 100ul of PBS. The infection period was run for 3 days, and the weight ofthe animals was monitored daily (we also extracted organs to evaluatebacterial loads in organs, results not shown). At that point the animalswere sacrificed. The output of the experiment was calculated as thepercentage body weight loss between day 3 and day 0 for every animal.See Tables 2-4.

TABLE 2 Multivalent vaccine experiments: Vaccinogen combinations AntigenTier 1 FtsL SEQ 5 SEQ10 SEQ19 Tier 2 Tier 3 YdiE DivlB DivlC SEQ24 YdiEDivlB DivlC FtsL YdiE DivlB DivlC FtsL PheP ✓ ✓ ✓ ✓ SEQ 1 ✓ ✓ SEQ 6 ✓ ✓SEQ 15 ✓ SEQ 20 ✓ ✓ ✓ YdiE ✓ SEQ 2 ✓ ✓ ✓ SEQ 7 ✓ ✓ SEQ 16 ✓ SEQ 21 ✓DivlB ✓ ✓ SEQ 3 ✓ SEQ 8 SEQ 17 ✓ SEQ 22 DivlC SEQ 4 SEQ 9 ✓ SEQ 18 SEQ23

TABLE 3 Body Weight of Immunized and Infected Mice % Body Weight LossControl Sample Experimental Samples I IV Adjuvant II III CombinationAnimal alone Antigen: Antigen: Antigen: Number (Freunds) RYdiE rDivlBrYdiE rDivlB 1 11.1 11.3 13.1 2.3 2 12.7 4.3 0.0 2.0 3 9.7 9.0 3.5 0.0 47.6 12.6 0.5 −4.3 5 9.4 1.0 7.1 −0.5 6 9.9 6.2 3.3 1.8 7 2.0 3.8 2.1 1.58 4.2 5.3 1.9 1.4 9 1.0 3.4 1.0 1.6 10 3.1 7.0 6.8 3.9 11 7.7 4.7 −2.5−0.5

TABLE 4 CFU in Kidneys of Immunized and Infected Mice Log10 CFUs inKidneys per animal Control Sample Experimental Samples I IV Adjuvant IIIII Combination Animal alone Antigen: Antigen: Antigen: Number (Freunds)rYdiE rDivlB rYdiE rDivlB 1 7.82 6.98 5.6 6.04 2 7.73 6.5 6.5 6.12 3 7.75.61 4.64 5.97 4 6.98 7.61 5.99 5.61 5 6.74 5.09 6.54 5.38 6 7.32 6.386.03 5.88 7 6.5 6.6 6.41 6.74 8 6.2 6.75 6.66 5.96 9 5.61 6.72 7.88 6.9810 6.06 6.63 6.63 6.68 11 7.04 6.45 4.74

What is claimed is:
 1. A composition, comprising two or more differentnucleic acid molecules each encoding an immunogenic staphylococcalextracellular domain polypeptide, wherein the nucleic acid molecules areselected from: (i) a nucleic acid molecule comprising or consisting of anucleotide sequence as represented in SEQ ID NO.:7; (ii) a nucleic acidmolecule comprising or consisting of a nucleotide sequence asrepresented in SEQ ID NO.:8; (iii) a nucleic acid molecule comprising orconsisting of a nucleotide sequence as represented in SEQ ID NO.:9; (iv)a nucleic acid molecule comprising or consisting of a nucleotidesequence as represented in SEQ ID NO.:10; (v) any one of the nucleicacids of (i) to (iv), wherein the nucleotide sequence is degenerate as aresult of the genetic code, provided that the degenerate nucleotidesequence encodes the same staphylococcal immunogenic extracellulardomain polypeptide as the nucleic acid molecules of (i) to (iv),respectively; (vi) any one of the nucleic acid molecules of (i) to (iv),wherein the nucleic acid molecule comprises or consists of a nucleotidesequence having at least 90% identity to the nucleotide sequence of thenucleic acid molecules of (i) to (iv), respectively; and (vii) a nucleicacid molecule, wherein the complementary strand of the nucleic acidmolecule hybridizes under stringent hybridization conditions to thenucleotide sequence as represented in SEQ ID NO.:7, 8, 9 or 10, andwherein the nucleic acid molecule encodes an immunogenic staphylococcalextracellular domain polypeptide.
 2. An immunogenic composition,comprising: (i) an immunogenic extracellular domain polypeptide, orimmunogenic fragment thereof, comprising or consisting of the amino acidsequence as represented in SEQ ID NO.:21, and an immunogenicextracellular domain polypeptide, or immunogenic fragment thereof,comprising or consisting of the amino acid sequence as represented inSEQ ID NO.:23; (ii) an immunogenic extracellular domain polypeptide, orimmunogenic fragment thereof, comprising or consisting of the amino acidsequence as represented in SEQ ID NO.:21, and an immunogenicextracellular domain polypeptide, or immunogenic fragment thereof,comprising or consisting of the amino acid sequence as represented inSEQ ID NO.:24; (iii) an immunogenic extracellular domain polypeptide, orimmunogenic fragment thereof, comprising or consisting of the amino acidsequence as represented in SEQ ID NO.:22, and an immunogenicextracellular domain polypeptide, or immunogenic fragment thereof,comprising or consisting of the amino acid sequence as represented inSEQ ID NO.:23; (iv) an immunogenic extracellular domain polypeptide, orimmunogenic fragment thereof, comprising or consisting of the amino acidsequence as represented in SEQ ID NO.:22, and an immunogenicextracellular domain polypeptide, or immunogenic fragment thereof,comprising or consisting of the amino acid sequence as represented inSEQ ID NO.:24; (v) an immunogenic extracellular domain polypeptide, orimmunogenic fragment thereof, comprising or consisting of the amino acidsequence as represented in SEQ ID NO.:23, and an immunogenicextracellular domain polypeptide, or immunogenic fragment thereof,comprising or consisting of the amino acid sequence as represented inSEQ ID NO.:24; (vi) an immunogenic extracellular domain polypeptide, orimmunogenic fragment thereof, comprising or consisting of the amino acidsequence as represented in SEQ ID NO.:21, an immunogenic extracellulardomain polypeptide, or immunogenic fragment thereof, comprising orconsisting of the amino acid sequence as represented in SEQ ID NO.:23,and an immunogenic extracellular domain polypeptide, or immunogenicfragment thereof, comprising or consisting of the amino acid sequence asrepresented in SEQ ID NO.:24; (vii) an immunogenic extracellular domainpolypeptide, or immunogenic fragment thereof, comprising or consistingof the amino acid sequence as represented in SEQ ID NO.:22, animmunogenic extracellular domain polypeptide, or immunogenic fragmentthereof, comprising or consisting of the amino acid sequence asrepresented in SEQ ID NO.:23, and an immunogenic extracellular domainpolypeptide, or immunogenic fragment thereof, comprising or consistingof the amino acid sequence as represented in SEQ ID NO.:24; or (viii)any one of the compositions of (i) to (vii), wherein the immunogenicextracellular domain polypeptide or immunogenic fragment thereof is animmunogenic extracellular domain polypeptide variant having at least 90%identity to the amino acid sequence presented in the corresponding SEQID NO.
 3. The immunogenic composition according to claim 2, comprising:(i) an immunogenic extracellular domain polypeptide comprising SEQ IDNO.:21, or an antigenic fragment thereof; and (ii) an immunogenicextracellular domain polypeptide comprising SEQ ID NO.:23, or anantigenic fragment thereof.
 4. The immunogenic composition according toclaim 2, comprising: (i) an immunogenic extracellular domain polypeptidecomprising SEQ ID NO.:21, or an antigenic fragment thereof; and (ii) animmunogenic extracellular domain polypeptide comprising SEQ ID NO.:24,or an antigenic fragment thereof.
 5. The immunogenic compositionaccording to claim 2, comprising: (i) an immunogenic extracellulardomain polypeptide comprising SEQ ID NO.:21, or an antigenic fragmentthereof; and (ii) an immunogenic extracellular domain polypeptidecomprising SEQ ID NO.:23, or an antigenic fragment thereof, and (iii) animmunogenic extracellular domain polypeptide comprising SEQ ID NO.:24,or an antigenic fragment thereof.
 6. The immunogenic compositionaccording to claim 2, comprising (i) an immunogenic extracellular domainpolypeptide comprising SEQ ID NO.:22, or an antigenic fragment thereof;and (ii) an immunogenic extracellular domain polypeptide comprising SEQID NO.:23, or an antigenic fragment thereof.
 7. The immunogeniccomposition according to claim 2, comprising: (i) an immunogenicextracellular domain polypeptide comprising SEQ ID NO.:22, or anantigenic fragment thereof; and (ii) an immunogenic extracellular domainpolypeptide comprising SEQ ID NO.:24, or an antigenic fragment thereof.8. The immunogenic composition according to claim 2, comprising: (i) animmunogenic extracellular domain polypeptide comprising SEQ ID NO.:22,or an antigenic fragment thereof; and (ii) an immunogenic extracellulardomain polypeptide comprising SEQ ID NO.:23, or an antigenic fragmentthereof, and (iii) an immunogenic extracellular domain polypeptidecomprising SEQ ID NO.:24, or an antigenic fragment thereof.
 9. Theimmunogenic composition according to claim 2, comprising: (i) animmunogenic extracellular domain polypeptide comprising SEQ ID NO.:23,or an antigenic fragment thereof; and (ii) an immunogenic extracellulardomain polypeptide comprising SEQ ID NO.:24, or an antigenic fragmentthereof.
 10. The immunogenic composition according to claim 2, furthercomprising a carrier, an adjuvant or both.
 11. The immunogeniccomposition according to claim 2, wherein the composition is a vaccine.12. The immunogenic composition according to claim 11, wherein thevaccine is adapted for administration as a nasal spray.
 13. Theimmunogenic composition according to claim 12, wherein the vaccine isprovided in an inhaler and delivered as an aerosol.
 14. A method oftreating a staphylococcal infection in an animal subject, comprisingadministering an effective amount of an immunogenic compositionaccording to claim
 1. 15. The method of claim 14, wherein theimmunogenic composition further comprises a carrier, an adjuvant orboth.
 16. The method of claim 15, wherein the adjuvant is aluminiumphosphate.
 17. The method of claim 14, wherein the immunogeniccomposition further comprises an adjuvant and the composition isformulated as a gel.
 18. The immunogenic composition according to claim14, wherein the immunogenic composition is administered as an aerosol ornasal spray.
 19. The immunogenic composition according to claim 14,wherein the staphylococcal infection is caused by a staphylococcalspecies selected from the group consisting of: S. epidermidis, S.aureus, S. hominis, S. haemolyticus, S. wameri, S. capitis, S.saccharolyticus, S. auricularis, S. simulans, S. saprophyticus, S.xylosus, S. hyicus, S. caprae, S. gallinarum, and S. intermedius. 20.The immunogenic composition according to claim 14, wherein the animalsubject is human, caprine or bovine.